Bottom Line:
Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism.CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation.The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

ABSTRACTSchnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

fig9: Proposed model for role of UBIAD1 in sterol-accelerated degradation of HMG CoA reductase.In sterol-deprived cells, both reductase and UBIAD1 localize to membranes of the ER. The intracellular accumulation of sterols in ER membranes triggers binding of reductase to Insigs, resulting in its ubiquitination by Insig-associated ubiquitin ligases gp78 and Trc8 and association with UBIAD1. Geranylgeraniol becomes phosphorylated to produce geranylgeranyl pyrophosphate, which enhances reductase degradation by binding to UBIAD1, causing its displacement from reductase-Insig. This displacement allows for transport of UBIAD1 to the Golgi and membrane extraction, cytosolic dislocation, and proteasomal degradation of reductase. We postulate that the SCD-associated N102S or G177R mutations in UBIAD1 abrogate binding of geranylgeranyl pyrophosphate. As a result, UBIAD1 (N102S) and (G177R) do not translocate to the Golgi and remain associated with reductase in the ER, thereby blocking its membrane extraction, cytosolic dislocation, and proteasomal degradation.DOI:http://dx.doi.org/10.7554/eLife.05560.019

Mentions:
Results of the current experiments form the basis for the model shown in Figure 9 that depicts the role of UBIAD1 in sterol-accelerated ERAD of reductase. As previously proposed (Morris et al., 2014), the reaction is initiated by accumulation of sterols, which triggers binding of Insigs to reductase and results in its gp78/Trc8-mediated ubiquitination (Song et al., 2005; Jo et al., 2011). We find in the current study that the oxysterol 25-HC and the 1,1-bisphosphonate ester Apomine, which mimics 25-HC in accelerating reductase ERAD (Roitelman et al., 2004; Sever et al., 2004; Nguyen et al., 2009), trigger binding of reductase to UBIAD1 (Figure 3B,C), a prenyltransferase that mediates synthesis of the vitamin K2 derivative MK-4 (see Figure 1). The sterol-induced binding of UBIAD1 to reductase appears to follow the action of Insigs as indicated by inhibition of the association by RNAi-mediated Insig knockdown (Figure 4A). In contrast to the action of sterols, the nonsterol isoprenoid geranylgeraniol, which augments sterol-accelerated reductase ERAD (Sever et al., 2003a), inhibits formation of the UBIAD1-reductase complex (Figure 5A and Figure 5—figure supplement 1B). Geranylgeraniol also regulates the subcellular localization of UBIAD1. The prenyltransferase primarily localizes to Golgi membranes when cells are cultured in sterol-replete FCS-containing medium (Figure 5C). The Golgi localization of UBIAD1 is disrupted when cells are switched to sterol-depleting medium containing LPPS and the reductase inhibitor compactin, which depletes cellular stores of nonsterol isoprenoids (Brown and Goldstein, 1980). Remarkably, Golgi localization of UBAID1 is restored by the addition of geranylgeraniol, but not of 25-HC or farnesol, to cells deprived of sterol and nonsterol isoprenoids (Figure 5C and Figure 5—figure supplements 2, 3). We conclude from these observations that geranylgeraniol-mediated disruption of the UBIAD1-reductase complex allows for the translocation of UBIAD1 from ER membranes to the Golgi and for membrane extraction, cytosolic dislocation and proteasomal degradation of reductase (Figure 9).10.7554/eLife.05560.019Figure 9.Proposed model for role of UBIAD1 in sterol-accelerated degradation of HMG CoA reductase.

fig9: Proposed model for role of UBIAD1 in sterol-accelerated degradation of HMG CoA reductase.In sterol-deprived cells, both reductase and UBIAD1 localize to membranes of the ER. The intracellular accumulation of sterols in ER membranes triggers binding of reductase to Insigs, resulting in its ubiquitination by Insig-associated ubiquitin ligases gp78 and Trc8 and association with UBIAD1. Geranylgeraniol becomes phosphorylated to produce geranylgeranyl pyrophosphate, which enhances reductase degradation by binding to UBIAD1, causing its displacement from reductase-Insig. This displacement allows for transport of UBIAD1 to the Golgi and membrane extraction, cytosolic dislocation, and proteasomal degradation of reductase. We postulate that the SCD-associated N102S or G177R mutations in UBIAD1 abrogate binding of geranylgeranyl pyrophosphate. As a result, UBIAD1 (N102S) and (G177R) do not translocate to the Golgi and remain associated with reductase in the ER, thereby blocking its membrane extraction, cytosolic dislocation, and proteasomal degradation.DOI:http://dx.doi.org/10.7554/eLife.05560.019

Mentions:
Results of the current experiments form the basis for the model shown in Figure 9 that depicts the role of UBIAD1 in sterol-accelerated ERAD of reductase. As previously proposed (Morris et al., 2014), the reaction is initiated by accumulation of sterols, which triggers binding of Insigs to reductase and results in its gp78/Trc8-mediated ubiquitination (Song et al., 2005; Jo et al., 2011). We find in the current study that the oxysterol 25-HC and the 1,1-bisphosphonate ester Apomine, which mimics 25-HC in accelerating reductase ERAD (Roitelman et al., 2004; Sever et al., 2004; Nguyen et al., 2009), trigger binding of reductase to UBIAD1 (Figure 3B,C), a prenyltransferase that mediates synthesis of the vitamin K2 derivative MK-4 (see Figure 1). The sterol-induced binding of UBIAD1 to reductase appears to follow the action of Insigs as indicated by inhibition of the association by RNAi-mediated Insig knockdown (Figure 4A). In contrast to the action of sterols, the nonsterol isoprenoid geranylgeraniol, which augments sterol-accelerated reductase ERAD (Sever et al., 2003a), inhibits formation of the UBIAD1-reductase complex (Figure 5A and Figure 5—figure supplement 1B). Geranylgeraniol also regulates the subcellular localization of UBIAD1. The prenyltransferase primarily localizes to Golgi membranes when cells are cultured in sterol-replete FCS-containing medium (Figure 5C). The Golgi localization of UBIAD1 is disrupted when cells are switched to sterol-depleting medium containing LPPS and the reductase inhibitor compactin, which depletes cellular stores of nonsterol isoprenoids (Brown and Goldstein, 1980). Remarkably, Golgi localization of UBAID1 is restored by the addition of geranylgeraniol, but not of 25-HC or farnesol, to cells deprived of sterol and nonsterol isoprenoids (Figure 5C and Figure 5—figure supplements 2, 3). We conclude from these observations that geranylgeraniol-mediated disruption of the UBIAD1-reductase complex allows for the translocation of UBIAD1 from ER membranes to the Golgi and for membrane extraction, cytosolic dislocation and proteasomal degradation of reductase (Figure 9).10.7554/eLife.05560.019Figure 9.Proposed model for role of UBIAD1 in sterol-accelerated degradation of HMG CoA reductase.

Bottom Line:
Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism.CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation.The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

ABSTRACTSchnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.